Biology Reference
In-Depth Information
A
B
C
D
E
F
G
H
I
RGC survival
% Innervation
Density of innervation
2.5
100000
60
2
Saline
rCNTF+CPT-cAMP
50
40
1.5
50000
30
1
20
0.5
10
0
0
0
Saline
rCNTF+
CPT-cAMP
Saline
rCNTF+
CPT-cAMP
Lateral SC
Mid SC
Medial SC
Figure 1.1 (A) Retinal whole mount showing surviving bIII-tubulin-positive retinal
ganglion cells (RGCs) and axons after contralateral 2 mm transection of optic tract
and intravitreal injections of rCNTF and CPT-cAMP. Note viability of both large and small
RGCs. For analysis of reinnervation, cholera toxin subunit B (CTB) was injected into the
eye and, 48 h later, rats were euthanased and transcardially perfused. Sections of
the superior colliculus (SC) were cut sagittally and stained for CTB using dia-
minobenzidine immunohistochemistry. Analysis was carried out in every third section
starting from the medial-most section. B
-
F, low power views (rostral to left) of sagittal
sections of postlesion SC stained for CTB-labeled retinal axons and terminals (B
-
C,
rCNTFþCPT-cAMP-injected animals; D
-
F, saline-injected animals). Grid in D used to
guide randomsampling for densitometry analysis. (G
-
I) histograms showingquantitative
assessment of RGC survival (G), percent reinnervation (H) and density of reinnervation in
medial, mid, and lateral SC (I). For % reinnervation, the total volume of the superficial
layers of the SC and the volume of tissue containing DAB-stained profiles in those layers
weremeasured using Image Pro at 40magnification. The volume of reinnervated tissue
was expressed as a % of total SC volume (H). For density of reinnervation, a grid was sup-
erimposed on the SC (D) and images captured using 100 lens under oil. In every third
section from medial to lateral SC, average pixel density was calculated in three grids
randomly sampled in rostral, mid, and caudal SC regions using Image Pro software (nine
samples per section). SC sections from control (no lesion) and saline-injected lesion
animals were always stained at the same time as experimental (rCNTFþCPT-cAMP)
sections. Settings for light intensity, gain, offset, andpinhole sizewere normalizedagainst
nonlesion control
sections and held constant
for all
sections
in lesioned
rCNTFþCPT-cAMP and saline-injected animals. Scale bars: A¼70 mm; B
-
F¼500 mm.
